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​Ongoing Research

DNA replication is a fundamental process in the life cycle of every living organism and its regulation is essential for the normal development of cells and their regulated proliferation. Regulation of chromosome replication is mediated by interactions of replication origins and other control elements in the DNA with their trans-acting proteins. Our studies focus on the mechanisms that control these interactions, and thereby, regulate the initiation of DNA replication in eukaryotes, using the replication of the kinetoplast DNA (kDNA) of trypanosomatids as an experimental model. Although kDNA is an extrachromosomal genome, its replication provides an attractive model for the study of several major aspects of chromosomal DNA replication, as the two systems share several major features in common, including the control of duplication of multiple replicons, and restriction of their replication to the S-phase of the cell cycle.
kDNA is a DNA network found in the single mitochondrion of trypanosomatids. The network consists of ~5,000 duplex DNA minicircles and ~25 maxicircles, which are interlocked into a disk-shape catenane. Two short sequences, a 12-mer "universal minicircle sequence" (UMS) and a hexamer, were conserved at the origins of the minicircle L and H strands (oriL and oriH), respectively. The minicircle origin binding protein, designated the universal minicircle sequence binding protein (UMSBP), which binds these sequences, is one of the major subjects of our recent research.
In earlier studies, we have described in C. fasciculata the single-stranded DNA binding protein UMSBP, that interacts specifically with the conserved UMS sequence at the oriL and with a 14-mer sequence (H14, including the conserved hexamer) at the oriH. UMSBP's encoding gene and genomic locus were cloned and analyzed and were found to be conserved in other trypanosomatid species. UMSBP is a 13.7 kDa protein, containing five CCHC-type zinc fingers, which oligomerize in solution. The protein was immunolocalized to two neighboring sites, in the kineto-flagellar zone (KFZ), in the same region as DNA primase, Pol I-type DNA polymerases and a center of minicircle replication intermediates. Structure-function analysis has revealed that UMSBP binds the DNA ligand as a monomer. The protein N-terminal domain, mediates its dimerization, while the C-terminal domain is involved in UMSBP-DNA interactions. Regulation of UMSBP binding at the minicircle replication origin seems to be mediated through two post translational modification pathways, redox signaling and protein phosphorylation. Both DNA binding and dimerization of UMSBP are sensitive to redox. Oxidation results in UMSBP dimerization, with concomitant inhibition of its DNA binding activity, while reduction yields monomers that are active in the binding of DNA. C. fasciculata tryparedoxin activates the binding of UMSBP to UMS DNA in vitro. An N-terminally truncated UMSBP mutant was apparently released from the redox effect, suggesting that UMSBP N-terminal domain plays a major role in the redox-mediated regulation of UMSBP. In addition to its control through redox signaling, UMSBP is phosphorylated in the cell at serine residues, through the action of a protein kinase C-type protein kinase.
Other aspects of our current research focus on the recognition of the minicircle replication origin by the replication machinery and the parameters that determined the choice of a functional origin; the assembly of the replication initiation complex; DNA helicases that are involved in kDNA replication and the parameters that characterize the movement of the minicircle replication fork.
Assigning functions to genes is one of the major challenges of the post-genomic era. Usually, functions are assigned based on similarity of the coding sequences to sequences of known genes, or by identification of transcriptional cis-regulatory elements that are known to be associated with specific pathways or conditions. In trypanosomatids, where regulation of gene expression takes place mainly at the post-transcriptional level, new approaches for function assignment are needed. In a recent study, that included a whole genome analysis of Leishmania major, we demonstrated the identification of novel S-phase expressed genes in Leishmania major, based on a post-transcriptional control element that was recognized in Crithidia fasciculata as involved in the cell cycle-dependent expression of several nuclear and mitochondrial S-phase expressed genes. Hypothesizing that a similar regulatory mechanism is manifested in L. major, we have applied a computational search for similar control elements in the genome of L. major. Our computational scan yielded 132 genes, 33% of which are homologues of known DNA metabolism genes and 63% lack any annotation. Experimental testing of seven of these genes revealed that their mRNAs cycle throughout the cell cycle, reaching a maximum level during S-phase or just prior to it. It is suggested that screening for post-transcriptional control elements associated with a specific function provides an efficient method for assigning functions to trypanosomatid genes.